RNA transformation vector

ABSTRACT

A +strand RNA viral transformation of host organisms with foreign RNA, and expression of said foreign RNA. The foreign RNA is inserted into an infective RNA viral segment containing replication elements, and allowed to infect the host organism. The invention is exemplified utiliing brome mosaic RNA modified to contain a gene coding for chloramphenicol acetyl transferase (CAT) in the transformation of barley protoplasts.

This is a continuation of application Ser. No. 08/212/330, filed Mar.14, 1994, now U.S. Pat. No. 5,500,360; which is a continuation of Ser.No. 07/916,799, filed Jul. 17, 1992, now abandoned; which is acontinuation of Ser. No. 07/368,939, filed Jun. 19, 1989, now abandoned;which is a continuation of Ser. No. 06/709,181, filed Mar. 7, 1985, nowabandoned.

FIELD OF THE INVENTION

This invention relates to the field of plant viruses, more particularlyto (+) strand RNA viruses of plants animals and bacteria, and tomodifications, made according to the teachings herein, which permitinsertion of an exogenous RNA segment into the viral genome. Theinserted segment can then be introduced into a host cell in order tomodify the cell, either genotypically or phenotypically. The inventionis exemplified by modifications of an RNA plant virus, brome mosaicvirus (BMV), which is infective for monocots.

BACKGROUND AND PRIOR ART

RNA viruses whose genome is composed of a single RNA strand capable ofreplication in the cytoplasm of a host by direct RNA replication arewide-spread, many varieties of which are known and which infect animals,plants and bacteria. Such viruses are sometimes termed "(+) strand RNAviruses" since the infective RNA strand, that normally foundencapsidated in the virus particle, is a messenger-sense strand, capableof being directly translated, and also capable of being replicated underthe proper conditions by a direct process of RNA replication. Virusesbelonging to this group include, but are not limited to, thepicornaviruses, the RNA bacteriophages, the comoviruses, and varioussingle component and multicomponent RNA viruses of plants. A partiallisting of such viruses would include polio virus, sindbis virus, Qβbacteriophage, tobacco mosaic virus, barley stripe mosaic virus, cow peamosaic virus, cucumber mosaic virus, alfalfa mosaic virus and bromemosaic virus. In some cases, the entire virus genome is contained withina single RNA molecule, while in other cases, most notably themulticomponent RNA plant viruses, the total genome of the virus consistsof two or more distinct RNA segments, each separately encapsidated. (Forgeneral review, see General Virology, S. Luria and J. Darnell; PlantVirology 2nd ed., R. E. F. Matthews, Academic Press (1981); and for ageneral review of (+) strand RNA replication, see Davies and Hull (1982)J. Gen. Virol. 61,1). Within the group there are wide variations incapsid morphology, coat proteins, genetic organization genome size.

Despite the well-documented diversity, recent studies have shownstriking similarities between the proteins which function in RNAreplication. Sequence homologies have been reported between the cowpeamosaic virus, poliovirus and foot-and-mouth disease virus (Franssen, H.(1984) EMBO Journal 3,855), between non-structural proteins encoded byalfalfa mosaic virus, brome mosaic virus and tobacco mosaic virus,Haseloff, J. et al. (1984), Proc. Nat. Acad. Sci. USA 81, 4358, andbetween the same proteins and proteins encoded by sindbis virus,Ahlquist, P. et al. (1985) J. Virol. 53, 536. Evidence of suchsubstantial homology in proteins related to the replication functionsindicate that the viruses share mechanistic similarities in theirreplication strategies and may actually be evolutionarily related. Inthe present invention, modifications to the genomic RNA of a (+) strandRNA virus are disclosed. The modified RNA is used to transfer a desiredRNA segment into a targeted host cell and to replicate that segment andexpress its function within the host cell. A virus known to berepresentative of the common replication functions of (+) strand RNAviruses was chosen to exemplify the present invention herein.

Brome mosaic virus (BMV) is one member of a class of plant virusescharacterized by a multipartite RNA genome. The genetic material of thevirus is RNA, and the total genetic information required for replicationand productive infection is divided into more than one discrete RNAmolecule. The class, termed multipartite RNA viruses herein, includes,besides BMV, such viruses as alfalfa mosaic virus (AMV), barley stripemosaic virus, cowpea mosaic virus, cucumber mosaic virus, and manyothers. Virus particles are generally composed of RNA encapsidated by aprotein coat. The separate RNA molecules which comprise the total genomeof a given multipartite virus are encapsidated in separate virusparticles, each of which has the same protein composition. Infection ofa host plant cell occurs when a virus particle containing each of theRNA components of the viral genome has infected the cell, for example byexposing a plant to a virus preparation containing a mixture of allnecessary viral components. Infection may also be achieved by exposing aplant cell or protoplast to a mixture of the RNA components. A subclassof the multipartite RNA viruses (termed subclass I herein) requires coatprotein in addition to viral RNA for replication and productiveinfection. AMV is an example of a subclass I multipartite virus. Anothersubclass (termed subclass II herein) does not require coat protein, thecomponent RNAs being both necessary and sufficient for replication andproductive infection. BMV belongs to subclass II. The BMV genome isdivided among three messenger-sense RNAs of 3.2, 2.8 and 2.1 kilobases(Ahlquist, P. et al. (1981) J. Mol. Biol. 153,23; Ahiquist, P., et al.(1984) J. Mol. Biol. 172,369). The term "messenger-sense" denotes thatthe viral RNAs can be directly translated to yield viral proteins,without the need for an intervening transcription step.

Complete cDNA copies of each of the three BMV genetic components havebeen cloned in a general transcription vector, pPM1, described byAhlquist, P. and Janda, M. (1984) Mol. Cell Biol. 4,2976. Three plasmidshave been selected, pB1PM18, pB2PM25 and pB3PM1 containing,respectively, cDNA copies of BMV-RNA1, BMV-RNA2 and BMV-RNA3. The threeplasmids constitute, as a set, the complete BMV genome.

DNA from each of the three BMV cDNA-containing plasmids can be cleavedat a unique EcoRI site. The linear DNA thus produced can be transcribedin vitro in a reaction catalyzed by RNA polymerase. A modified λ P_(R)promoter in the transcription vector, pPM1, allows RNA synthesis toinitiate exactly at the 5' terminus of each BMV sequence, andtranscription continues to the end of the DNA template, adding 6-7nonviral nucleotides at the 3' ends of the transcripts. Whentranscription is carried out in the presence of a synthetic capstructure, m⁷ GpppG, as described by Contreras, R., et al. (1982)Nucleic Acids Res. 10,6353, RNA transcripts are produced with the samecapped 5' ends as authentic BMV RNA's. These RNAs are active messengersin in vitro translation systems and direct production of proteins withthe same electrophoretic mobilities as those translated from authenticBMV RNAS.

SUMMARY OF THE INVENTION

For the sake of brevity, the term "RNA virus" is used herein to mean (+)strand replicating RNA viruses.

The invention is based on the discovery that an RNA of the genome of anRNA virus can be modified to include an exogenous RNA segment and thatthe modified RNA can be introduced into a host cell, replicated thereinand can express the exogenous RNA segment. The recipient cell is therebyphenotypically transformed and may contribute to a genotypicallytransformed organism, as well. Phenotypically transformed cells can bemodified in vivo, in planta, in tissue culture, in cell culture or inthe form of protoplasts. The exemplified embodiment of the invention isuseful for producing phenotypically transformed plants under fieldconditions or greenhouse growth. Traits desirable for introduction inthis manner include, but are not limited to, pest resistance, pathogenresistance, herbicide tolerance or resistance, modified growth habit andmodified metabolic characteristics, such as the production ofcommercially useful peptides or pharmaceuticals in plants. Themodifications can be applied at any time during the growth cycle,depending on the need for the trait. For example, resistance to a pestcould be conferred only if the crop were at risk for that pest, and atthe time when the crop was most likely to be affected by the pest. Othertraits can be used to enhance secondary properties, for example toincrease the protein content of post-harvest forage. Any plant varietysusceptible to infection by a multipartite RNA virus can bephenotypically transformed. The choice of virus and the details ofmodification will be matters of choice depending on parameters known andunderstood by those of ordinary skill in the art. Other uses for cellsand organisms phenotypically or genotypically modified by means of amodified RNA derived from an RNA virus will be readily apparent to thoseskilled in the art, given a wide range of RNA viruses to modify and awide range of susceptible host cell types. Other uses for transformedanimal cells, bacterial cells and the like can be readily envisioned.For example, bacterial cells susceptible to Qβ phage can be grown inculture to desired cell density, infected with a modified Qβ phagecarrying a desired gene and thereby caused to express large quantitiesof a desired protein within a short time period.

Generally, the steps of a process for phenotypically transforming a cellor organism are: forming a full-length cDNA transcript of the virus RNA,or of each RNA component if the RNA virus is multipartite; cloning eachcDNA in a transcription vector; modifying the cDNA of at least one ofthe RNA components by inserting a non-viral DNA segment in a region ableto tolerate such insertion without disrupting RNA replication thereof;transcribing the modified cDNA, or, in the case of a multipartite virus,transcribing each cDNA corresponding to an RNA component of themultipartite virus; substituting the modified cDNA for its unmodifiedcounterpart in the transcription reaction; infecting virus-susceptibleprotoplasts, cells, tissues or whole organisms with transcribed RNA, ora mixture of RNAs, either in solution or encapsidated, of each viralcomponent including the modified RNA comprising messenger-sense RNAcontaining an exogenous RNA segment. From this point, the steps to befollowed will vary, depending on the type of material infected and theroute of infection. Protoplasts, cells and tissues of plants can bepropagated vegetatively, regenerated to yield whole plants by means ofany technique suitable to the particular plant variety infected, andtransplanted to the field. Whole plants can be infected in situ.Infected plants and plant cells can produce many copies per cell of themodified viral RNA containing the exogenous RNA segment. If desired andif suitably inserted, by means of principles and processes known in theart, the exogenous RNA segment can be caused to carry out a functionwithin the cell. Such a function could be a coding function, translatedwithin the cell to yield a desired peptide or protein, or it could be aregulatory function, increasing, decreasing, turning on or off theexpression of certain genes within the cell. Any function which asegment of RNA is capable of providing can, in principle, be expressedwithin the cell. The exogenous RNA segment thus expressed confers a newphenotypic trait to the transformed organism, plant, cells, protoplastsor tissues.

The invention is exemplified herein by the modification of BMV RNA tocontain a structural gene encoding chloramphenicol acetyl transferase(CAT) and the phenotypic modification of barley protoplasts therewith,yielding protoplasts synthesizing CAT. The data presented herein arebelieved to represent the first instance of phenotypic modification of acell by means of a modified RNA of an RNA virus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a map of EcoRI-cleaved pB3M1.

FIG. 2 shows a 21 nucleotide mismateh primer used to prime synthesis ofDNA from M13/B3ES1.

FIG. 3 shows the insertion of the CAT gene in in-frame linkage with theinitiation codon of th BMV coat protein gene.

FIG. 4 shows the insertion of the CAT gene at the SalI site of pBΩP1.

FIG. 5 shows file construction of pB3DCP.

FIG. 6 (Parts a and b) shows the production of CAT by protoplastsinoculated with tanscripts constructed according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

In order to facilitate understanding of the invention, certain termsused throughout are herein defined.

RNA virus--The term as used herein means a virus whose genome is RNA insingle-stranded form, the single strand being a (+) strand, ormessenger-sense strand. Replication of the viral (+) strand in avirus-infected cell occurs by a process of direct RNA replication and istherefore distinguishable from the replication mechanism of retroviruseswhich undergo an intermediate step of reverse transcription in the hostcell.

Cis-acting replication element--This term denotes that portion of theRNA genome of an RNA virus which must be present in cis, that is,present as part of each viral strand as a necessary condition forreplication. Virus replication presumably depends upon the existence ofone or more trans (diffusible) elements which interact with thecis-acting element to carry out RNA replication. While trans-actingelements are necessary for replication, they need not be present orcoded for on the modified RNA provided they are made available withinthe infected cell by some other means. For example, in the case of amulitpartite RNA virus, the trans-acting functions may be provided byother, unmodified components of the viral genome used to transform thecells simultaneously with the modified RNA. The target cell may also bemodified in a previous step to provide constitutive expression of thetrans-acting functions. The cis-acting replication element is composedof one or more segments of viral RNA which must be present on any RNAmolecule that is to be replicated within a host cell by RNA replication.The segment will most likely be the 5' terminal portion of the viral RNAmolecule, and may include other portions as well. The cis-acting elementis therefore defined in functional terms: any modification whichdestroys the ability of the RNA to replicate in a cell known to containthe requisite trans-acting elements, is deemed to be a modification inthe cis-acting replication element. Conversely, any modification, suchas an insertion in a sequence region which is able to tolerate suchinsertion without disrupting replication, is a modification outside thecis-acting replication element. As is demonstrated herein, using theexample of BMV, substantial portions of an RNA virus molecule may bemodified, by deletion, insertion, or by a combination of deletion andinsertion, without disrupting replication.

The term "derived from" is used to identify the viral source of an RNAsegment which comprises part of the modified RNA. For example, for themodified RNAs described herein, substantial portions thereof are derivedfrom BMV. The manner of deriving, whether by direct recombination at theRNA level, by transcription or by reverse transcription does not matterfor the purpose of the invention. Indeed, it is contemplated thatmodifications may be made within the cis-acting replication element andelsewhere for example to modify the rate or amount of replication thatis obtained. In the case of modified RNAs exemplified herein, atranscription vector was employed which preserved the exact 5' terminalnucleotide sequence of viral RNA. However the use of such a vector intranscribing viral RNA from cDNA is not considered essential to theinvention, although it will be preferred if preservation of the exactnucleotide sequence at the 5' end is desired. An RNA segment which hasbeen derived from a given source virus may, but need not be, identicalin sequence to that segment as-it exists in the virus. It will beunderstood that a cis-acting replicating element derived from a givenRNA virus may have minor modifications in the nucleotide sequencethereof without substantially interfering with RNA replication.

Exogenous RNA segment is a term used to describe a segment of RNA to beinserted into the virus RNA to be modified, the source of the exogenousRNA segment being different from the RNA virus itself. The source may beanother virus, a living organism such as a plant, animal, bacteria,virus or-fungus; the exogenous RNA may be a chemically synthesized RNAor it may be a combination of the foregoing. The exogenous RNA segmentmay provide any function which is appropriate and known to be providedby an RNA segment. Such functions include, but are not limited to, acoding function in which the RNA acts as a messenger RNA encoding asequence which, translated by the host cell, results in synthesis of apeptide or protein having useful or desired properties; the RNA segmentmay also be structural, as for example in ribosomal RNA, it may beregulatory, as for example with small nuclear RNA's or anti-sense RNA,or it may be catalytic. A particularly interesting function is providedby antisense RNA, sometimes termed (-) strand RNA, which is in fact asequence complementary to another RNA sequence present in the targetcell which can, through complementary base pairing, bind to and inhibitthe function of the RNA in the target cell.

Various aspects of the stages outlined in the Summary section can bemodified as needed, depending upon specific aspects of the virusselected as the transforming agent and of the RNA segment to beinserted. For example, if the inserted gene is in the form ofmessenger-sense RNA to be directly translated by the transformed cell,the gene must be free of intervening, nontranslated sequences, such asintrons. On the other hand, the inserted gene need not be a naturallyoccurring gene, but may be modified, a composite of more than one codingsegment, or it may encode more than one protein. The RNA may also bemodified by combining insertions and deletions in order to control thetotal length or other properties of the modified RNA molecule. Asdemonstrated in Example 5, a substantial portion of the RNA3 of BMV canbe deleted without significantly effecting its replication in cellscontaining normal RNA1 and RNA2. The inserted non-viral gene may beeither prokaryotic or eukaryotic in origin as long as it is in a formwhich can be directly translated by the translation machinery of therecipient cell. Eukaryotic genes containing introns within the codingsequence must therefore be inserted in the form of a cDNA copy of theeukaryotic messenger RNA encoding the gene. The inserted gene maycontain its own translation start signals, for example, a ribosomalbinding site and start (AUG) codon, or it may be inserted in a mannerwhich takes advantage of one or more of these components preexisting inthe viral RNA to be modified. Certain structural constraints must beobserved to preserve correct translation of the inserted sequence,according to principles well understood in the art. For example, if itis intended that the exogenous coding segment is to be combined with anendogenous coding segment, the coding sequence to be inserted must beinserted in reading frame phase therewith and in the same translationaldirection. The term "non-viral" is used herein in a special sense toinclude any RNA segment which is not normally contained within the viruswhose modification is exploited for effecting gene transfer and istherefore used synonymously with "exogenous". Therefore, a gene derivedfrom a different virus species than that modified is included within themeaning of the terms "non-viral" and "exogenous" for the purposes ofdescribing the invention. For example, a non-viral gene as the term isused herein could include a gene derived from a bacterial virus, ananimal virus, or a plant virus of a type distinguishable from the virusmodified to effect transformation. In addition, a non-viral gene may bea structural gene derived from any prokaryotic or eukaryotic organism.It will be understood by those ordinarily skilled in the art that theremay exist certain genes whose transfer does not result in obviousphenotypic modification of the recipient cell. Such may occur, forexample, if the translation product of the nonviral gene is toxic to thehost cell, is degraded or processed in a matter which renders itnon-functional or possesses structural features which render itimpossible for the host cell to translate in sufficient quantities toconfer a detectable phenotype on the transformed cells. However, theinvention does not depend upon any specific property of an RNA segmentor gene being transferred. Therefore, the possible existence of RNAsegments or genes which fail to confer a readily observable phenotypictrait on recipient cells or plants is irrelevant to the invention and inany case will be readily recognizable by those of ordinary skill in theart without undue experimentation.

An exogenous RNA segment may be inserted at any convenient insertionsite in any of the cDNA sequences corresponding to a viral RNA, orcomponent RNA of a multipartite RNA virus, provided the insertion doesnot disrupt a sequence essential for replication of the RNA within thehost cell. For example, for a virus whose coat protein is not essentialfor replication, an exogenous RNA segment may be inserted within orsubstituted for the region which normally codes for coat protein. Asdesired, regions which contribute to undesirable host cell responses maybe deleted or inactivated, provided such changes do not adversely effectthe ability of the RNA to be replicated in the host cell. For manysingle component and multipartite RNA viruses, a reduction in the rateof normal RNA replication is tolerable and will in some instances bepreferred, since the amount of RNA produced in a normal infection ismore than enough to saturate the ribosomes of the transformed cell.

The transformation process itself can be carried out by any meanswhereby RNA can be introduced into cells, whole plants, plant tissues orprotoplasts. Host cells can be infected by the RNA alone, orencapsidated in a virus particle, except that the modified viral RNAcontaining a non-viral RNA segment is substituted for its counterpart ina normal infection. Any other suitable means for introducing RNA intotarget cells such as microinjection may be used. In some cases it may bepreferable to include all of the normal components in addition to themodified component. More than one component may be modified in themixture of transforming components. It will be understood that theamounts of each infecting component must be sufficient to insure that anadequate number of cells receive at least one of each component in themixture. Other variables of the infection process, such as pretreatmentof the recipients, addition of components to enhance the efficiency ofinfection, use of encapsidated or unencapsidated RNA, are matters ofchoice which those of ordinary skill in the art will be able tomanipulate to achieve desired transformation efficiency in a givensituation. For instance, the choice of multipartite plant RNA virus tobe modified to effect gene transfer in a given plant variety will dependupon known host range properties of multipartite RNA viruses. Forexample, BMV infects a variety of grasses and their related domesticatedrelatives including barley, wheat and maize. Plant cells which areinfected in culture will normally remain transformed as the cells growand divide since the RNA components are able to replicate and thusbecome distributed to daughter cells upon cell division. Plantsregenerated from phenotypically modified cells, tissues or protoplastsremain phenotypically modified. Similarly, plants transformed asseedlings remain transformed during growth. Timing of application of thetransforming components will be governed by the result which is intendedand by variations in susceptibility to the transforming componentsduring various stages of plant growth.

Many plant RNA viruses are seed transmitted from one generation to thenext. This property can be exploited to effect genotypic transformationof a plant. That is to say, the modified RNA remains transmissible fromone generation to the next, most likely by replication in the cytoplasm,and thereby becomes transmissible from one generation to the next, justas seed-borne virus infections are transmitted from one generation tothe next.

The following examples illustrate the principles of the invention asapplied to modification of BMV RNA3 and the use of modified BMV RNA3containing a gene coding for chloramphenicol acetyl transferase (CAT) inthe phenotypic transformation of barley protoplasts. For convenience,any modification to a viral RNA which includes the insertion of anonviral ribonucleotide sequence, whether or not combined with adeletion of viral RNA will be designated by a prime symbol following thenumber designating the RNA. For example, modified RNA3 is termed RNA3',or more generally, RNAn is designated RNAn'.

The following examples utilize many techniques well known and accessibleto those skilled in the arts of molecular biology, cloning, plant cellbiology, plant virology and plant tissue culture. Such methods are fullydescribed in one or more of the cited references if not described indetail herein. Unless specified otherwise, enzymes were obtained fromcommercial sources and were used according to the vendor'srecommendations or other variations known to the art. Reagents, buffersand culture conditions and reaction conditions for various enzymecatalyzed reactions are also known to those in the art. Reference workscontaining such standard techniques include the following: R. Wu, ed.(1979) Meth. Enzymol. 68; R. Wu et al., eds. (1983) Meth. Enzymol. 100,101; L. Grossman and K. Moldave, eds. (1980) Meth. Enzymol. 65; J. H.Miller (1972) Experiments in Molecular Genetics; R. Davis et al. (1980)Advanced Bacterial Genetics; R. F. Schleif and P. C. Wensink (1982)Practical Methods in Molecular Biology; and T. Manniatis et al. (1982)Molecular Cloning.

Textual use of the name of a restriction endonuclease in isolation,e.g., "BclI" refers to use of that enzyme in an enzymatic digestion,except in a diagram where it can refer to the site of a sequencesusceptible to action of that enzyme, e.g., a restriction site. In thetext, restriction sites are indicated by the additional use of the word"site", e.g., "BclI site". The additional use of the word "fragment",e.g., "BclI fragment", indicates a linear double-stranded DNA moleculehaving ends generated by action of the named enzyme (e.g., a restrictionfragment). A phrase such as "BclI/SmaI fragment" indicates that therestriction fragment was generated by the action of two differentenzymes, here BclI and SmaI, the two ends resulting from the action ofdifferent enzymes. Note that the ends will have the characteristics ofbeing either sticky (i.e., having a single strand of protrusion capableof base-pairing with a complementary single-stranded oligonucleotide) orblunt (i.e., having no single-stranded protrusion) and that thespecificity of a sticky end will be determined by the sequence ofnucleotides comprising the single-stranded protrusion which in turn isdetermined by the specificity of the enzyme which produces it.

All plasmids are designated by a sequence of letters and numbersprefaced by a lower case "p", for example, pPM1. Clones of complete BMVcDNA inserted in pPM1 are named by the format pBxPMy, where x equals 1,2 or 3 designating the BMV component cloned (i.e., from RNA1, 2 or 3)and y is an arbitrary isolate number. Thus, the set of three plasmids,pB1PM18, pB2PM25 and pB3PM1 contains complete cDNA copies of BMV RNAs 1,2 and 3, respectively, and represent, as a set, the complete BMV genome.Certain steps of cloning, selection and vector increase employed strainsof E. coli. While the strains used herein have been designated, thereare many equivalent strains available to the public which may beemployed. The use of a particular microorganism as a substitute for astrain designated herein is a matter of routine choice available tothose of ordinary skill in the art, according to well-known principles.

EXAMPLE 1 Infectivity of transcribed BMV-cDNA

In vitro Transcription. Transcription reactions contained 25 mMTris-HCl, pH 8.0/5 mM MgCl₂ /150 mM NaCl/1 mM dithiothreitol/200 μM eachrATP, rCTP, and rUTP/25 μM rGTP/500 μM m₇ GpppG (P-LBiochemicals)/plasmid DNA (0.1 μg/μl) Escherichia coli RNA polymerase(0.05 units/μl) (Promega Biotec, Madison, Wis.). Reactions wereincubated 30 minutes at 37° C., by which time the GTP was nearlyexhausted. Additional rGTP was added to 25 μM and incubation continued afurther 30 minutes. For uncapped transcripts, m⁷ GpppG was deleted, GTPwas increased to 200 μM, the concentrations of DNA and polymerase weredoubled, and incubation was carried out for 1 hour. Reactions werestopped by addition of EDTA to 10 mM and either diluted directly ininoculation buffer or phenol-extracted before nucleic acid recovery byethanol precipitation. In most experiments, plasmids representing allthree BMV components were pooled and cleaved at unique EcoRI sites 3base pairs past the 3' terminus of each BMV sequence beforetranscription. FIG. 1 shows a map of EcoRI-cleaved pB3M1. The maps forpPM1 containing cDNA of RNA1 or RNA2 are the same, except that theregion labeled "BMV-cDNA" is cDNA of RNA-1 or RNA-2.

Infectivity Testing. Seven-day-old barley seedlings (Hordeum vulgare L.cv. Morex) were dusted with carborundum powder and inoculated witheither virion RNA or in vitro transcription mixes in 50 mM Tris PO₄, pH8.0/250 mM NaCl/5 mM EDTA/Bentonite (5 mg/ml) (5); 15-30 plants in asingle 14-cm-diameter pot were treated with the same inoculum, using10-30 μl per plant. Plants were scored for the presence of mosaicsymptoms 7-14 days after inoculation.

BMV Isolation. Fourteen days after inoculation, virus was isolated frombarley plants as described by Shih, et al. (1972) J. Mol. Biol. 64,353,with the substitution of chloroform for carbon tetrachloride and asecond polyethylene glycol precipitation for differentialcentrifugation. Viral RNA was isolated by phenol extraction and ethanolprecipitation.

Infectivity Testing of BMV cDNA Clones and Their in vitro Transcripts.Cloning of complete cDNA copies of all three BMV genetic components in ageneral transcription vector, pPM1, has been described by Ahlquist, P.and Janda, M. (1984) Mol. Cell. Biol. 4,2876. DNA from such clones canbe cleaved with EcoRI (FIG. 1) and transcribed in vitro in the presenceof a synthetic cap structure to produce complete RNA copies of the BMVcomponents that have the same capped 5' ends as authentic BMV RNA's, andan additional 6-7 non-viral nucleotides at their 3' ends.

To test the infectivity of these cloned DNAs and their transcripts,three plasmids, pB1PM18, pB2PM25, and pB3PM1, were selected. Theselected clones contain cDNA copies of BMV RNAs 1, 2, and 3,respectively, and represent, as a set, the complete BMV genome. Thenatural isolate of BMV propagated in our laboratory is referred to byits usual designation of Russian strain. Mixtures of the EcoRI-cut M1plasmids and their capped transcription products were inoculated ontobarley plants in parallel with untranscribed DNA from the same plasmids.As judged by the production of normal viral symptoms, the transcribedplasmid mixture was infectious, while the untranscribed plasmid mixturewas not (Table 1).

                  TABLE 1                                                         ______________________________________                                        Comparison of infectivity of EcoRI-cut M1 plasmids; transcribed               EcoR1-cut M1 plasmids, and Russian strain BMV virion RNA's over a             range of inoculum concentrations.                                                                      Plants with                                          Pot No.    Inoculum, ng/μl                                                                          symptoms/total                                       ______________________________________                                                 EcoRI-cut pB1PM18,                                                            pB2PM25; pB3PM1                                                      1          100           0/21                                                 2          10            0/23                                                 3          1             0/22                                                          Transcribed EcoRI-cut                                                         pB1PM18, pB2PM25,                                                             pB3PM1                                                               4          40            19/23                                                5          4             7/20                                                 6          0.4           0/21                                                          Russian strain BMV                                                            virion RNA                                                           7          10            21/22                                                8          1             14/21                                                9          0.1            2/21                                                         Mock-inoculated                                                      10         0             0/22                                                 ______________________________________                                    

In vitro transcription yields approximately 3 BMV transcripts perplasmid (Ahlquist and Janda, 1984). Total BMV transcript content of theinocula for pots 4-6 is thus approximately 75, 7.5, and 0.75 ng/μl,respectively.

The effects of various alterations to the transcription protocol wereexamined to more clearly characterize the infectious entity observed inplasmid transcription mixes. Infectivity required transcription ofclones representing all three BMV genetic components. Moreover,infectivity was sensitive to HinfI before or to RNase A aftertranscription, but it was not significantly affected by RNase A beforeor HinfI after transcription. HinfI cleaves at 8 sites within pPM1 andat 15, 10, and 12 sites within BMV 1, 2, and 3 cDNAs, respectively.These results confirm that the observed infectivity arises from the invitro transcripts rather than directly from their DNA templates. Inaddition, when plasmids were either not cut or were cut with PstI beforetranscription (cleaving 2.7 kilobases rather than 7 bases downstream ofthe cDNA end), infection was not observed, suggesting that infectivityis affected by the structure of the transcript 3' end. Finally, if thecap analog was omitted during in vitro transcription, no infection wasdetected, even if inoculum concentration was increased 20-fold.

Infectivity of RNA transcribed in vitro from EcoRI-cut M1 plasmids wasclearly lower than that of authentic BMV RNA. The number of infectedplants produced from a given weight of in vitro-transcribed RNA wassimilar to that produced from 1/10th that weight of authentic BMV RNA(Table 1). The presence of the plasmid DNA template in the inoculum wasnot responsible for this effect, as addition of the same plasmid DNA toauthentic BMV RNA did not affect its infectivity.

Correlation of Symptomology with BMV Replication. To establish that suchsymptoms accurately reflect BMV replication, several molecular testswere applied. Nitrocellulose dot blots of total RNA (described byGarger, S. J. et al. (1983) Plant Mol. Biol. Reporter 1,21) extractedfrom leaves of symptomexpressing and symptomless plants inoculated witheither authentic BMV RNA or in vitro BMV transcripts were probed with ³²P-labeled cloned BMV cDNA. In all cases, symptom-expressing leavesshowed a positive hybridization response, and in all cases but one,symptomless leaves gave a negative response. The one exception was froma plant that had been inoculated with in vitro transcripts and showed novisible symptoms but gave a positive hybridization signal.

Virus isolated from plants infected with cDNA transcripts isserologically identical to Russian strain BMV in double-diffusion testswith anti-BMV antisera. Phenol extraction of BMV isolated fromtranscript-infected plants releases four RNAs that comigrate withRussian strain virion RNAs, hybridize to BMV-specific DNA probes, andare highly infectious in subsequent inoculations. Therefore,multipartite RNA plant virus infection can be derived solely fromappropriately cloned viral cDNA by means of a simple transcription step.

EXAMPLE 2 Construction and replication of a specific deletion in the BMVcoat gene

In the following example, reference may be made to FIG. 1 for locationof the relevant restriction sites.

Plasmid pB3PM1 DNA (Ahlquist, P. and Janda, M. (1984)) was cleaved withSalI and XbaI and treated with the Klenow fragment of DNA polymerase Ito generate blunt ends (Maniatis et al. (1982) Molecular Cloning: ALaboratory Manual. Cold Spring Harbor). The approximately 5.2 kbfragment was isolated from a low melting point (LMP) agarose gel (Sangeret al. (1980) J. Mol. Biol. 143,161), recircularized by treatment withT4 DNA ligase, and transformed into competent E. coli JM101. RF DNA fromselected ampicillin resistant transformants was digested simultaneouslywith SalI and EcoRI to confirm regeneration of the SalI site anddeletion of the desired fragment. A single tested clone, designatedpB3DCP10, having the region of the coat gene from SalI to SbaI deleted,was selected for further work.

EcoRI-digested pB3DCP10 was transcribed under capping conditions(Ahlquist and Janda, 1984) along with EcoRI-digested pB1PM18 and pB2PM25(Ahlquist and Janda, 1984), and the transcripts were separated from theplasmid DNA templates by LiCl precipitation (Baltimore (1966) J. Mol.Biol. 18, 421). Barley protoplasts were prepared as described byLoesch-Fries and Hall (1980) J. Gen. Virol. 47, 323. and inoculated asdescribed by Samac et al. (1983) Virology 131, 455, with the transcriptsand incubated in the presence of ³ H!uridine. Total nucleic acids wereextracted and analyzed on acrylamide-agarose gels as described byLoesch-Fries and Hall, 1980. The deleted RNA3 derived from pB3DCP10 wasfound to both replicate and generate a deleted version of subgenomicRNA4. (RNA4 is a subgenomic fragment of RNA3 produced during infection).This example demonstrates that a substantial portion of RNA3 encodingcoat protein can be deleted, without preventing replication of viralRNA's.

EXAMPLE 3 Insertion of a PstI site at the 3' cDNA end of plasmid pB3PM1

Construction and use of transcribable BMV cDNA clones has been describedbefore (Ahlquist et al., 1984a, 1984b). To define the transcript 3' end,the originally described plasmids are first linearized beforetranscription by cleavage of an EcoRI site just outside the 3' end ofBMV cDNA. However, such EcoRI cleavage results in addition of 6-7nonviral nucleotides to the transcript and is inconvenient fortranscription of BMV-linked foreign sequences which contain EcoRIsite(s). To deal with both of these problems, a PstI site, i.e., anucleotide sequence including a sequence recognized and cleaved by PstIendonuclease, was inserted immediately adjacent to the BMV cDNA inpB3PM2 to provide an alternate cleavage site. The steps in theconstruction can be followed by referring to FIG. 2. DNA sequences shownin FIG. 2 are plus (+) strands only, defined as equivalent (notcomplementary) to the RNA sequence of BMV-RNA.

Insertion of this PstI site was generally similar to the previouslydescribed insertion of a SmaI site adjacent to the lambda P_(R) promoter(Ahlquist and Janda, 1984). First the 0.9 kb SalI-EcoRI fragment ofpB3PM1 (FIG. 1) was isolated from a low-melting point agarose gel andsubcloned into SalI EcoRI cleaved M13mp9. Colorless recombinant plaqueswere selected on X-gal/IPTG plates and the insertion of BMV sequencesverified by dideoxynucleotide sequencing (Biggen et al. (1983) Proc.Nat. Acad. Sci. USA 80, 3963). A single clone, designated M13/B3ES1, wasselected for further work. A 21 nucleotide mismatch primer (FIG. 2) waschemically synthesized and purified and used to prime synthesis of ³²P-labeled DNA from M13/B3ES1 ssDNA. After synthesis, the DNA was cleavedwith AvaI at a site in the M13 vector distal to the primer and the majorlabelled DNA fragment, containing the mismatch primer at its 5' end andBMV3 sequences interior, was purified on an alkaline agarose low meltingpoint gel (Maniatis et al., 1982). A second strand of DNA was primedwith a lac reverse primer (Ahlquist and Janda, 1984), the ds syntheticDNA cleaved with XbaI and the approximately 0.36 kb dsDNA fragment,containing the mismatch primer linked to 3' BMV RNA3 sequences, isolatedfrom a low melting point agarose gel. This fragment was then subclonedinto XbaI-SmaI cut M13mp19. Colorless recombinant plaques were selectedon X-gal/IPTG plates and the correct linkage of the PstI site to BMVcDNA confirmed by dideoxy sequencing. The 0.36 kb XbaI EcoRI fragmentfrom a selected M13 clone was recloned between the XbaI and SalI sitesof Pb3PM1, creating plasmid pB3ΩP1. The sequence of RNA transcribed fromPstI-cleaved pB3ΩP1 will be identical to that of BMV RNA3 except thatthe 3'-terminal A will be omitted.

EXAMPLE 4 Insertion of a bacterial chloramphenicol resistance gene in aBMV RNA3 derivative and expression of a functional protein in barleycells

Plasmid pB3ΩP1 (Example 3) was cleaved with SalI and XbaI to delete mostof the coat protein gene except for seven nucleotides at the beginningof the coat protein coding sequence including the AUG start codon,treated with the Klenow fragment of DNA polymerase I to produce bluntends and the resulting larger DNA fragment isolated from a low meltingpoint agarose gel. Plasmid pBR325 (Bolivar, 1978) was digested withTaqI, treated with Klenow polymerase and the 780 bp fragment containingthe chloramphenicol acyl transferase (CAT) gene was isolated. The 780 bpfragment isolated in this manner contained the entire CAT gene togetherwith a short segment of pBR325 flanking the 5' end of the CAT genecoding sequence. The larger pB3ΩP1 fragment and a three-fold molarexcess of the CAT fragment were ligated with T4 DNA ligase andtransformed into E. coli JM101 cells. Plasmid DNA from selectedampicillin-resistant transformants was screened by double digestion withEcoRI and PstI and gel electrophoresis to confirm insertion of the CATgene and to determine its orientation with respect to BMV3 cDNAsequences. One plasmid, pB3CA42, containing the CAT gene codingsequences in the same orientation as the BMV3 coding sequences wasselected for further work along with a plasmid, pB3CA52, with the CATgene in the reverse orientation. Insertion of the CAT gene in thepositive orientation, as in pB3CA42, results in in-frame linkage of theCAT coding sequences with the initiation codon of the BMV coat gene(FIG. 3). Translation from the coat AUG would result in production of afusion protein bearing 12 additional amino acids before the start of thenative CAT gene product.

In a similar construction, diagrammed in FIG. 4, the same CAT fragmentwas inserted at the SalI site of pB3Ω3 by SalI digestion followed byblunt ending with Klenow polymerase and ligation with DNA ligase. Twoclones differing in the orientation of the CAT gene were isolated,pB3CA31 with the CAT gene coding sequence oriented backwards from thedirection of transcription, and pB3CA21 with the CAT gene codingsequence oriented in the same direction as that of transcription. FIG. 4also shows the nucleotide sequence in the region of the junction pointbetween BMV-derived and bacterial-derived sequences, for pB3CA21. As acontrol, SalI/XbaI deleted pB3 without an insertion was constructed,designated pB3DCP, as shown in FIG. 5. The sequence in the region of thesubgenomic transcription start site and religation site is also shown inFIG. 5. As a further control, the CAT coding sequence was deleted fromplasmid pB3CA42 (FIG. 3) by cleaving with SalI, filling out the recessed3' ends with Klenow DNA polymerase and deoxynucleotides, and religatingthe resultant blunt ends.

PstI-cut pB3CA42 DNA and EcoRI-cut pB1PM18 and pB2PM25 DNAs weretranscribed, LiCl-precipitated and used to inoculate protoplasts(Example 1). After 22 hours incubation protoplasts were lysed byfreezing and thawing and were found to contain CAT activity as assayedby standard methods (Herrera-Estrella et al. (1983) Nature 303, 209;Shaw, (1975) Methods Enzymol. 53, 737). Cell lysates were incubated with¹⁴ C! chloramphenicol and, following the published procedure, silica gelthin layer plates separating reactants and products wereautoradiographed. The results are shown in FIG. 6. Lanes marked Cm wereloaded with ¹⁴ C! chloramphenicol only. CAT activity in other reactionsis indicated by the appearance of acetylated chloramphenicol formsmarked 1A-Cm (1-acetate) and 3A-Cm (3-acetate) in addition to the nativeform marked Cm. The lanes marked CAT-mi and CAT show the productsproduced by authentic bacterial CAT in the presence of extracts frommock-inoculated protoplasts or buffer only. Panel A shows the productsproduced by extracts obtained from protoplasts inoculated withtranscripts from pB1PM18 and pB2PM25, together with pB3CA21 (lanedesignated CA 21), pB3CA31 (lane designated CA 31), pB3CA42 (lanedesignated CA 42), pB3CA52 (lane designated CA 52), pB3CA61 (lanedesignated CA 61) or pB3PM1 (lane designated 3-1). In panel B theproducts obtained from extracts of protoplasts inoculated with variouscombinations of pB1PM18 (designated 1), pB2PM25 (designated 2); pB3PM1(designated 3), and pB3CA42 (designated 3°) are shown. In paralleltests, mockinoculated protoplasts and protoplasts inoculated withtranscripts from EcoRI-cut pB1PM18, EcoRI-cut pB2PM25 and eitherEcoRI-cut pB3PM1 or PstI-cut pB3CA52 showed no detectable CAT activity.The results shown in FIG. 6 demonstrate phenotypic transformation of thecells and further demonstrate that an RNA-3' containing an insertednonviral coding segment, under appropriate conditions of infection, caneffect such transformation. Only the combination of 1°+2°+3° providesexpression of the CAT gene, showing that this expression is dependent onviral RNA replication.

EXAMPLE 5 Bal 31 Deletions in Plasmid pB3PM1

Plasmid pB3PM1 DNA was cleaved with ClaI, treated with T4 DNA polymeraseto produce blunt ends, and ligated to phosphorylated 12 bp syntheticBamHI linkers (Maniatis et al., 1982). After phenol/chloroformextraction and ethanol precipitation, the DNA was cleaved with 40 unitsBamHI per μg linker for 16 hours at 37° C. After electrophoresis on 1%(w/v) low-melting point agarose the major ethidium bromide-staining bandof DNA was eluted (Sanger et al., 1980) and recircularized by treatmentwith T4 DNA ligase at approximately 2 ng DNA/μl reaction, andtransformed into competent E. coli JM101. RF DNA from randomly selectedampicillin-resistant transformants was digested simultaneously withBamHI and EcoRI and screened by gel electrophoresis to confirm thepresence of the BamHI linker at the desired point. A single clone,designated pB3C49, was selected for further work.

12 μg of ClaI-cleaved pB3PM1 DNA was treated with 12 units of Bal 31 atroom temperature in a 180 μl reaction (Guo et al. (1983) Nucleic AcidsRes. 11, 2237). 30 μl aliquots were removed 2, 4, 6, 8, 10 and 12minutes after enzyme addition. Nuclease digestion in each aliquot wasterminated by addition of 25 μl of 40 mM EDTA and two successivephenol/chloroform extractions. The aliquots were pooled and the DNAprecipitated with ethanol. The DNA was treated with the Klenow fragmentof DNA polymerase I to generate blunt ends, and 12 bp synthetic BamHIlinkers were added (Maniatis et al, 1982). After phenol/chloroformextraction and ethanol precipitation, the DNA was treated with 50 unitsBamHI/μg linker and 2 units PstI/μg plasmid for 16 hours at 37° C.Products were run on a low melting point agarose gel and the high MWfraction containing the approximately 4.2 kb ClaI/PstI fragment ofpB3PM1 and its Bal 31-deleted, linker-ligated products was eluted andmixed with a molar excess of the approximately 1.5 kb PstI-BamHIfragment of pB3C49. After ligation, DNA was transformed into competentE. coli JM101 cells. RF DNA was prepared from randomly-selectedampicillin-resistant transformants and was screened by double digestionwith BamHI and EcoRI followed by agarose gel electrophoresis. Plasmidswith deletions extending a variety of distances from the initial ClaIsite, within the 3a gene (FIG. 1), of pB3PM1 toward the EcoRI site wereselected using this data. Selected plasmids were cleaved with EcoRI andtranscribed (Example 1) and the transcripts used to infect barleyprotoplasts in the presence of transcripts from EcoRI-cut plasmidspB1PM18 and pB2PM25.

Using similar techniques a BamHI linker was inserted in the SacI site ofpB3PM1 and two further Bal 31 deletion libraries were constructed, onewith deletions extending 5' to the Sacd site and one with deletions 3'to the SacI site. Transcripts from selected EcoRI-cut plasmids weretested in the presence of transcripts from EcoRI-cut pB1PM18 and pB3PM25in the barley protoplast system. Transcripts from pB3PM1 derivativeswith linker insertions in either the ClaI and SacI sites, and fromderivatives with deletions extending for up to several hundred basesfrom either site were found to replicate under such conditions.Substantial deletions within the 3a gene and the coat protein gene cantherefore be made, at least several hundred bases from either the ClaIsite of the SacI site, without preventing replication of the deletedRNA. Such deletions provide room for large insertions while stillstaying within the size constraints for packaging replicated RNA3' intovirus particles. The remaining portion of RNA, derived from RNA3,contains a cis-acting replication element of BMV RNA. Although the 3Agene and coat gene were not required for RNA replication or forexpression of the inserted CAT gene under the conditions of infectionused in the example, either or both of these genes could, under otherconditions, provide important secondary functions, for example, bypromoting systemic infection during transfer of whole plants. Wheredeletion is not desired but the length of the modified RNA exceeds thepackaging constraints of the icosahedral BMV capsid, it may be possibleto provide for expression of the coat protein of a rod-shaped virus forencapsidating the modified RNA.

Discussion and Conclusions

The foregoing examples demonstrate that substantial modifications, bothdeletions and insertions, can be made in an RNA component of amultipartite RNA virus without preventing replication of viral RNA underappropriate conditions of infection. Genetic material inserted within aregion of an RNA virus that is nonessential for RNA replication istranslatable. In the case of BMV, substantial portions of RNA3 can bedeleted without loss of the ability to replicate. Therefore any geneinserted within a nonessential region of an RNA component of an RNAmultipartite virus can be translated in the transformed cell, providedthe gene has appropriate ribosome binding and translation initiationsignals at its 5' end. These signals can be provided by the virus or bythe insert and the means for making translatable constructions is withinthe scope of capability of those ordinarily skilled in the art.

While the foregoing principles were illustrated in the case of BMV RNA3,it is apparent that any component of any RNA virus is a candidate formodifications of the type illustrated. For example, an exogenous RNAsegment could be inserted at any site of BMV RNA 1 or 2 which does notresult in loss of ability to replicate. Similarly, the RNA components ofother RNA viruses can be similarly manipulated, provided the insertionsand/or deletions employed do not prevent replication of viral RNA. Thetwo operating principles which permit the modification of a viral RNAcomponent to make it a vector for carrying translatable genetic materialinto a host cell are: (1) cloning a cDNA of the RNA component into atranscription vector capable of transcribing replicatable RNA from viralcDNA, and (2) the identification of a region in one of the viralcomponents that is nonessential for replication, into which a structuralgene can be inserted. The modified RNA will therefore contain, at aminimum, a cis-acting replication element derived from an RNA virus andan inserted exogenous RNA segment. Further modifications andimprovements following and embodying the teachings and disclosuresherein are deemed to be within the scope of the invention, as set forthin the appended claims.

We claim:
 1. An RNA molecule comprising a cis-acting replication elementderived from a (+) strand RNA plant virus capable of replication in aplant cell and further comprising an exogenous RNA segment capable ofexpressing its function in a host cell, said exogenous RNA segment beinglocated in a region of said RNA molecule able to tolerate said segmentwithout disrupting RNA replication of said RNA molecule in the presenceof trans-acting replication elements in said host cell.
 2. The RNA ofclaim 1, wherein the exogenous RNA segment codes for a peptide orprotein.
 3. The RNA of claim 1, wherein the exogenous RNA segmentcomprises antisense RNA.
 4. The RNA of claim 1, wherein the exogenousRNA segment comprises structural RNA.
 5. The RNA of claim 1, wherein theRNA segment comprises a regulatory RNA.
 6. The RNA of claim 1, whereinthe exogenous RNA segment comprises RNA having catalytic properties. 7.The RNA of claim 1, wherein the cis-acting replication element isderived from a multipartite plant virus.
 8. The RNA of claim 1, whereinthe cis-acting replication element is derived from tobacco mosaic virus.9. The RNA of claim 1, wherein the cis-acting replication element isderived from alfalfa mosaic virus.
 10. The RNA of claim 1, wherein thecis-acting replication element is derived from brome mosaic virus. 11.The RNA molecule of claim 1, encapsidated with a viral coat protein. 12.A DNA transcription vector comprising cDNA having one strandcomplementary to an RNA molecule comprising a cis-acting replicationelement derived from a (+) strand RNA plant virus capable of replicationin a plant cell and further comprising an exogenous RNA segment capableof expressing its function in a host cell, said exogenous RNA segmentbeing located in a region of said RNA molecule able to tolerate saidsegment without disrupting RNA replication of said RNA molecule in thepresence of trans-acting replication elements in said host cell.
 13. TheDNA transcription vector of claim 12, wherein one strand of the cDNAthereof is complementary to RNA coding for a nonviral protein orpeptide.
 14. The DNA transcription vector of claim 12, wherein onestrand of the cDNA thereof is complementary to RNA having a regulatory,structural, or catalytic property.
 15. The DNA transcription vector ofclaim 12, wherein one strand of the cDNA thereof is complementary to anRNA segment derived from a multipartite plant virus.
 16. The DNAtranscription vector of claim 12, wherein one strand of the cDNA thereofis complementary to an RNA segment derived from a tobacco mosaic virus.17. The DNA transcription vector of claim 12, wherein one strand of thecDNA thereof is complementary to an RNA segment derived from an alfalfamosaic virus.
 18. The DNA transcription vector of claim 12, wherein onestrand of the cDNA thereof is complementary to an RNA segment derivedfrom a brome mosaic virus.
 19. A method of modifying a host cell,genotypically or phenotypically, which method comprises introducing intothe cell an RNA molecule comprising a cis-acting replication elementderived from a (+) strand RNA plant virus capable of replication in aplant cell and further comprising an exogenous RNA segment capable ofexpressing its function in a host cell, said exogenous RNA segment beinglocated in a region of said RNA molecule able to tolerate said segmentwithout disrupting RNA replication of said RNA molecule in the presenceof transacting replication elements in said host cell, whereby theexogenous RNA segment confers a detectable trait in the host cell,thereby modifying said host cell.
 20. The method of claim 19, whereinthe exogenous RNA molecule codes for a peptide or protein.
 21. Themethod of claim 19, wherein the exogenous RNA segment comprisesantisense RNA.
 22. The method of claim 19, wherein the exogenous RNAsegment comprises structural RNA.
 23. The method of claim 19, whereinthe exogenous RNA segment comprises a regulatory RNA.
 24. The method ofclaim 19, wherein the exogenous RNA segment comprises RNA havingcatalytic properties.
 25. The method of claim 19, wherein the cis-actingreplication element is derived from a multipartite plant virus.
 26. Themethod of claim 19, wherein the cis-acting replication element isderived from tobacco mosaic virus.
 27. The method of claim 19, whereinthe cis-acting replication element is derived from alfalfa mosaic virus.28. The method of claim 19, wherein the cis-acting replication elementis derived from brome mosaic virus.
 29. The method of claim 19, whereinthe host cell is a plant cell.
 30. The method of claim 19, wherein thehost cell is a monocot plant cell.